|Gulati Sakhuja, Anju|
|Liu, Hsing Yeh|
Submitted to: Journal of Virological Methods
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 4/16/2009
Publication Date: 9/1/2009
Citation: Gulati Sakhuja, A.N., Sears, J.L., Nunez, A., Liu, H. 2009. PRODUCTION OF POLYCLONAL ANTIBODIES AGAINST PELARGONIUM ZONATE SPOT VIRUS COAT PROTEIN EXPRESSED IN ESCHERICHIA COLI AND APPLICATION FOR IMMUNODIAGNOSIS. Journal of Virological Methods. 160: 29-37.
Interpretive Summary: Pelargonium zonate spot virus (PZSV) is a recently identified virus infecting tomato plants in the United States. To develop serological diagnostic tools for the detection of this virus, the production of good quality antibodies is necessity. The coat protein (CP) gene of PZSV California isolate was amplified by RT-PCR. The amplicon was cloned into a bacterial expression vector (pTriEX-4 Ek/LIC) and sequenced to confirm the integrity of the ORF with N-terminal fusion tags. The plasmid pTriEX-4-PZSV-CP was transformed into Escherichia coli Rosetta 2(DE3)pLacI and the recombinant PZSV-CP was expressed as a fusion protein containing N-terminal hexa-histidine and S tags. Expressed PZSV-CP was purified under denaturing conditions by affinity chromatography yielding 3 mg refolded protein per 200 ml of bacterial culture, and used as an antigen for raising PZSV-CP antiserum in rabbits. Specificity of the antiserum to PZSV was shown by Western blot and ELISA. When used in Western blot analysis, the antiserum produced a specific reaction to the recombinant protein as well as to the viral coat protein of PZSV. The antiserum was successfully used in I-ELISA at dilutions of up to 1:16000 to detect PZSV in infected leaf samples. Direct ELISA was successful only with denatured antigens. The polyclonal antiserum was also able to detect PZSV infecting various plant species by ELISA as well as by Western blots. This is the first report on production of polyclonal antiserum against recombinant coat protein of PZSV and its use for detection and diagnosis of virus using serological methods.
Technical Abstract: Pelargonium zonate spot virus (PZSV), a new emerging disease on tomato in the United States, has been classified as the first member of new proposed genus, Anulavirus, within the family Bromoviridae and characterized as having unstable virions with weakly immunogenic properties. To develop serological method for detection for PZSV, which allow growers and researchers to identify this new tomato disease, is crucial to the tomato industry. It leads the way for increased vigilance towards the new tomato disease in the field and to the prevention of this disease before it reaches epidemic level. Advances have made it possible to use recombinant fusion proteins as antigens to raise diagnostic antibodies for plant viruses. In contrast to the conventional method of antigen preparation e.g. virion purification following virus propagation in a greenhouse, the recombinant approach is advantageous as it does not encounter the problems associated with virus purification and maintenance of live virus culture, and is fast and economical. This study addressed the possibility of using in vitro expressed PZSV coat protein as antigen for the production of PZSV specific antiserum and the use of prepared antiserum for ELISA and Western blotting.